Anti-replicative recombinant 5S rRNA molecules can modulate the mtDNA heteroplasmy in a glucose-dependent manner.

There is plenty of literature suggesting the existence of mitochondrial RNA import in metazoa (Kim et al. 2017). Personally, I am not so convinced by this and it seems only some unicellular eukaryotes and plants have mitochondrial RNA import (Schneider 2011). There was recently an excellent review from Gammage et al. where they discussed the possibility of mitochondrial RNA import in metazoan (Gammage et al. 2018).

From the point of view of mitochondrial RNA import, one of the most hotly debated RNAs is 5S rRNA, a well-known structural component of the cytosolic ribosome. Only some single cell eukaryotes still have it in their mitochondrial DNA (mtDNA) (Janouškovec et al. 2017) but other have lost it. Recent structures of the mitochondrial ribosome showed that it doesn’t possess a 5S rRNA (Greber et al. 2015) but the 5S rRNA has actually been replaced by mtDNA encoded tRNA, the type of which depends on the species in question (Rorbach et al. 2016). So, from the point-of-view of the mitochondrial ribosome, there is no reason to import 5S rRNA.

I collected below (most) of the publications studying 5S rRNA import into mitochondria.

Publication	Method	Result
Yoshionari et al. 1994	Northern using mitoplasts	5S rRNA in mitoplasts is protected by RNAase
Magalhães et al. 1998	Northern and RT-PCR of mitochondrial RNA	5S rRNA in mitoplasts is protected by RNAase
Entelis et al. 2001	Northern of mitochondrial RNA	Different version of 5S rRNA imported with different efficiencies
Smirnov et al. 2008	Northern of mitochondrial RNA	Different version of 5S rRNA imported with different efficiencies
Smirnov et al. 2010	Northern of mitochondrial RNA	5S rRNA has mitochondrial import factors
Smirnov et al. 2011	Northern of mitochondrial RNA	More 5S rRNA import into mitochondria in the presence of pre-mRpL18
Mercer et al. 2011	RNAseq (mitochondria vs mitoplasts)	5S rRNA mildly enriched in mitoplasts vs mitochondria
Comte et al. 2013	Northern of mitochondrial RNA	5S rRNA in mitoplasts is protected by RNAase
Towheed et al. 2014	Northern of mitochondrial RNA	5S rRNA in mitoplasts is protected by RNAase and not imported upon membrane depolarization (FCCP)
Loutre et al. 2018	Northern of mitochondrial RNA	5S rRNA enriched in mitoplasts

I won’t to go to the specifics of each paper but when going through all of them I would have liked to see a few important controls:

• Verification of the mitoplast (mitochondria without the outer membrane) purity on western using antibodies against mitochondrial outer membrane, intermembrane space, inner membrane and matrix proteins.
• Show that the cytosolic ribosomes are not anymore attached to the mitochondrial outer membrane.
• Show that the enriched mitochondria are not contaminated by microsomes or endoplasmic reticulum.
• A RNA control which is not imported into mitochondria but would be digested under the experimental RNAse treatment conditions.
• How the ectopic expression of 5S rRNA or its derivatives affects cytosolic ribosome assembly/function and how much this contributing to mitochondrial changes.

The fact that almost all published mitochondrial 5S rRNA studies come from one lab using always the same approach make me a bit wary. The one published study using RNAseq suggested that 5S rRNA is enriched in mitochondria (Mercer et al. 2011) but some unpublished work from Vamsi Mootha’s lab using similar approach suggests 5S rRNA is just a persistent contaminant in mitochondrial preparations (Wolf 2014).

There was recently a publication suggesting that modified 5S rRNA can targeted into mitochondria and function as an anti-replicative molecule to stop the replication of mtDNA molecule carrying a deletions (Loutre et al. 2018). Figure 3 of this paper immediately raises some concerns as one of the negative control, the cytosolic 5.8S rRNA is also among the mitochondrial RNAs. The authors conclude this amount of contamination to be negligible. Next the authors established cell lines with stable expression of 5S rRNA and its variants. It seems weird to me that they never tested how this ectopic expression affects the main function of 5S rRNA, that is the cytosolic translation. I’m just worried that all the effects they detect are secondary to the changes in cytosolic translation.

These cell lines expressing 5S rRNA had also a heteroplasmic mtDNA deletion and the authors wanted to see whether 5S rRNA expression would affect the heteroplasmy levels of this deletion. Well, 5S rRNA and its derivatives had no effect on the heteroplasmy level of mtDNA deletion in these cell lines under normal glucose but showed some changes in glucose-free media. It should be pointed out though, that they carried out multiple comparisons using Student’s t-test so I wouldn’t trust the results.

Some cell clones with stronger ectopic expression of 5S RNA variants did show decrease in the mtDNA deletion levels. However, this could be also caused by the fact that cytosolic translation is affecting thereby affecting the whole cellular function. Again, they did multiple comparisons using Student’s t-test to analyze these results.

All in all, I find the evidence for mitochondrial 5S rRNA weak at best and people should start controlling better how ectopic 5S rRNA expression affects cytosolic translation. Otherwise everything they see might just be secondary to these cytosolic effects.

Reference:

Comte C, Tonin Y, Heckel-Mager AM, Boucheham A, Smirnov A, Auré K, Lombès A, Martin RP, Entelis N, Tarassov I. Mitochondrial targeting of recombinant RNAs modulates the level of a heteroplasmic mutation in human mitochondrial DNA associated with Kearns Sayre Syndrome. Nucleic Acids Res. 2013. PMID: 23087375

Entelis NS, Kolesnikova OA, Dogan S, Martin RP, Tarassov IA. 5 S rRNA and tRNA import into human mitochondria. Comparison of in vitro requirements. J Biol Chem. 2001. PMID: 11551911

Gammage PA, Moraes CT, Minczuk M. Mitochondrial Genome Engineering: The Revolution May Not Be CRISPR-Ized. Trends Genet. 2017. PMID: 29179920

Greber BJ, Bieri P, Leibundgut M, Leitner A2 Aebersold R, Boehringer D, Ban N. Ribosome. The complete structure of the 55S mammalian mitochondrial ribosome. Science. 2015. PMID: 25837512

Kim KM, Noh JH, Abdelmohsen K, Gorospe M. Mitochondrial noncoding RNA transport. BMB Rep. 2017. PMID: 28115039

Loutre R, Heckel AM, Jeandard D, Tarassov I, Entelis N. Anti-replicative recombinant 5S rRNA molecules can modulate the mtDNA heteroplasmy in a glucose-dependent manner. PLoS One. 2018. PMID: 29912984

Magalhães PJ, Andreu AL, Schon EA. Evidence for the presence of 5S rRNA in mammalian mitochondria. Mol Biol Cell. 1998. PMID: 9725900

Mercer TR, Neph S, Dinger ME, Crawford J, Smith MA, Shearwood AM, Haugen E, Bracken CP, Rackham O, Stamatoyannopoulos JA, Filipovska A, Mattick JS. The human mitochondrial transcriptome. Cell. 2011. PMID: 21854988

Rorbach J, Gao F, Powell CA, D'Souza A, Lightowlers RN, Minczuk M, Chrzanowska-Lightowlers ZM. Human mitochondrial ribosomes can switch their structural RNA composition. Proc Natl Acad Sci U S A. 2016. PMID: 27729525

Schneider A. Mitochondrial tRNA import and its consequences for mitochondrial translation. Annu Rev Biochem. 2011. PMID: 21417719

Smirnov A, Tarassov I, Mager-Heckel AM, Letzelter M, Martin RP, Krasheninnikov IA, Entelis N. Two distinct structural elements of 5S rRNA are needed for its import into human mitochondria. RNA. 2008. PMID: 18314502

Smirnov A, Comte C, Mager-Heckel AM, Addis V, Krasheninnikov IA, Martin RP, Entelis N, Tarassov I. Mitochondrial enzyme rhodanese is essential for 5 S ribosomal RNA import into human mitochondria. J Biol Chem. 2010. PMID: 20663881

Smirnov A, Entelis N, Martin RP, Tarassov I. Biological significance of 5S rRNA import into human mitochondria: role of ribosomal protein MRP-L18. Genes Dev. 2011. PMID: 21685364

Towheed A, Markantone DM, Crain AT, Celotto AM, Palladino MJ. Small mitochondrial-targeted RNAs modulate endogenous mitochondrial protein expression in vivo. Neurobiol Dis. 2014. PMID: 24807207

Wolf AR. Leveraging genomic approaches to characterize mitochondrial RNA biology. Doctoral dissertation, Harvard University.

Yoshionari S, Koike T, Yokogawa T, Nishikawa K, Ueda T, Miura K, Watanabe K. Existence of nuclear-encoded 5S-rRNA in bovine mitochondria. FEBS Lett. 1994. PMID: 7508404

The mitochondrial DNA polymerase gamma degrades linear DNA fragments precluding the formation of deletions.

The mitochondrial DNA polymerase gamma degrades linear DNA fragments precluding the formation of deletions.

I posted recently how two labs have published data showing that the mitochondrial DNA  (mtDNA) polymerase gamma (POLGA) would degrade linear mtDNA fragments (Peeva et al. 2018) or mtDNA under starvation (Medeiros et al. 2018). Now a third group published similar results and they come from the lab of Carlos Moraes so you know are going to enjoy it (Nissanka et al. 2018).

In this study they used both mouse embryonic fibroblasts (MEFs) and actual mice. First, they expressed mitochondrially targeted restriction enzymes to produce linear mtDNA fragments and followed how quickly these are degraded in WT MEFs and mtDNA mutator MEFs. Clearly, MEFs expressing the exonuclease-deficient POLGA were not efficient in removing the linear mtDNA fragment which is a similar result to the Peeva et al. study.

Previously, Medeiros et al. and Peeva et al studied the degradation of mtDNA in yeast and cell culture, respectively, but it was unclear whether this would also take place in vivo. Moraes lab is rather experienced in introducing enzymes (restriction enzymes and mitoTALENs) into mitochondria in mice and as could be expected they introduced these mitochondrially targeted restriction enzymes into mice using adenovirus to study mtDNA degradation. As a result, it seems that POLGA is participating to the degradation of linear mtDNA fragments also in vivo.

Nissanka et al. also assessed whether the presence of linear mtDNA leads to mtDNA rearrangements such as circular mtDNA molecules with deletions. This seems to indeed be the case and it would be interesting to know whether these rearrangements would also take place in patients carrying pathogenic mutations in the replication machinery proteins. These results also suggest that mitochondrial zinc fingers (mtZFN) and mitoTALENs (Gammage et al. 2017) might have some unintended consequences. Both of these approaches are based on cutting the mtDNA molecules carrying a pathogenic mutation leading to the degradation of the molecule. In the ideal case, the loss of these pathogenic molecules would be replaced by the replication of the WT mtDNA molecules. Based on the results of Nissanka et al. in the non-ideal case the presence of these linear molecules could increase the amount of mtDNA rearrangements.

References:

Medeiros TC, Thomas RL, Ghillebert R, Graef M. Autophagy balances mtDNA synthesis and degradation by DNA polymerase POLG during starvation. J Cell Biol. 2018. PMID: 29519802

Nissanka N, Bacman SR, Plastini MJ, Moraes CT. The mitochondrial DNA polymerase gamma degrades linear DNA fragments precluding the formation of deletions. Nat Commun. 2018. PMID: 29950568

Gammage PA, Moraes CT, Minczuk M. Mitochondrial Genome Engineering: The Revolution May Not Be CRISPR-Ized. Trends Genet. 2017. PMID: 29179920

Peeva V, Blei D, Trombly G, Corsi S, Szukszto MJ, Rebelo-Guiomar P, Gammage PA, Kudin AP, Becker C, Altmüller J, Minczuk M, Zsurka G, Kunz WS. Linear mitochondrial DNA is rapidly degraded by components of the replication machinery. Nat Commun. 2018. PMID: 29712893

Replication machinery degrading linear mitochondrial DNA

Linear mitochondrial DNA (mtDNA) molecules with deletions are a curious case. To my knowledge, they are only present at high levels in a few genetic models, including the mtDNA mutator mouse(Trifunovic et al. 2004, Bailey et al. 2009), mtDNA mutator flies (Bratic et al. 2015), patients carrying mutations in mitochondrial genome maintenance exonuclease 1 (MGME1)(Nicholls et al. 2014) and MGME1 knockout mouse (Matic et al. 2018).

In the case of mtDNA mutator mouse, the level of this linear mtDNA with deletion remains stable with time (Kukat et al. 2009). Additionally, it has been shown using mitochondrially targeted restriction enzymes that linear mtDNA is rapidly degraded (Bayona-Bafaluy et al. 2005). Therefore the question remains, who is degrading these mtDNA fragments?

Moretton et al. tried to answer this question by linearizing mtDNA with mitochondrial targeted restriction enzyme followed by systematic knockdown of exonucleases known/suggested to be in mitochondria (Moretton et al. 2017). These exonucleases included ExoG, EndoG, MGME1, DNA2 and FEN1. EXD2 was not tested, but that was later shown to be localized on the mitochondrial outer membrane (Hensen et al. 2018). Perhaps surprisingly, none of the exonucleases studied by Moretton et al. seemed to participate in degradation of linear mtDNA fragments.

There is still at least one exonuclease that Moretton et al. did not study, which is the exonuclease activity of mitochondrial DNA polymerase (POLG). Indeed, in the absence of nucleotides POLG can engage in so called exonuclease mode where it starts degrading the primer in 3’-5’ direction (Bratic et al. 2015). A recent study in fruit flies tried to answer the question what enzyme degrades mtDNA in fly sperm (Yu et al. 2017). Yu et al. concluded that DmPOLG is involved in this degradation but it takes place independent of its exonuclease activity. I was not convinced by this paper because they saw mtDNA replication in punctae without DmPOLG-GFP and more importantly all the models were based on knocking down various proteins of the minimal mitochondrial replisome with variable efficiencies. Also, when I read this paper I was aware of some in-house work taking place in the group of Martin Graef.

Medeiros et al. showed in yeast that under starvation there is a strong mtDNA depletion if autophagy is inhibited and that this depletion can be rescued by increasing nucleoside pools through supplementation or genetically (Medeiros et al. 2018). Therefore, the authors hypothesized that under starvation the autophagy-deficient cells might have changes in nucleotide pools causing MIP1 (yeast POLG) to enter the above mentioned exonuclease mode and degrade mtDNA. Using an exonuclease-deficient POLG the authors showed that indeed it seems to be POLG which is degrading mtDNA.

The most recent paper on the subject comes from Peeva et al. (Peeva et al. 2018). Peeva et al. tested multiple exonucleases (EXOG, APEX2, ENDOG, FEN1, DNA2, MRE11, RBBP8, MGME1 and POLG) to see whether the knockout or knockdown of these enzymes would affect the degradation of linear mtDNA molecules. Somewhat similar to Medeiros et al., Peeva et al. also suggest that POLG together with MGME1 and TWINKLE would be degrading linear mtDNA.

So there it is. Both in yeast and human cells mtDNA seems to be degraded by the exonuclease activity of POLG. It would be interesting to see how the yeast mtDNA actually looks like at the molecular level when degraded as yeasts do not possess MGME1. One could therefore predict that these degraded molecules would not have blunt ends similarly to the results from humans where POLG is suggested to degrade mtDNA to the 3’-5’ direction and MGME1 to the 5’-3’ direction. It is still open question what controls this degradation, if it is controlled to begin with. Both Medeiros et al. and Peeva et al. hypothesize that low nucleotide pools might be a controlling factor. Although this hypothesis would make sense in the case of yeast under starvation and inhibited autophagy, I don’t see how this would work with these human cells under normal culture conditions. Clearly just linearizing mtDNA is sufficient to promote mtDNA degradation without manipulation of the dNTP pools.

Some open questions for the future:

• Is this kind of degradation present also in more in vivo conditions or whether it is only present in somewhat artificial conditions where most of mtDNA is linearized by restriction enzymes.
• What, if any, is controlling the balance between mtDNA synthesis and degradation?
• Is there a specific endonuclease cleaving the yeast mtDNA before POLG can start degrading mtDNA?
• How is mtDNA degraded in organisms without MGME1?

Perhaps this is a stretch but in Peeva et al. study the authors showed how in WT cells linear mtDNA is degraded piece by piece. Where mitophagy selective for damaged mtDNA to exist, which I think it does not (Kauppila et al. 2017), one would not expect to see these slowly degrading molecules but instead a bulk disappearance of linear mtDNA fragments.

References:

Bailey LJ, Cluett TJ, Reyes A, Prolla TA, Poulton J, Leeuwenburgh C, Holt IJ. Mice expressing an error-prone DNA polymerase in mitochondria display elevated replication pausing and chromosomal breakage at fragile sites of mitochondrial DNA. Nucleic Acids Res. 2009. PMID: 19244310

Bayona-Bafaluy MP, Blits B, Battersby BJ, Shoubridge EA, Moraes CT. Rapid directional shift of mitochondrial DNA heteroplasmy in animal tissues by a mitochondrially targeted restriction endonuclease. Proc Natl Acad Sci U S A. 2005. PMID: 16179392

Bratic A, Kauppila TE, Macao B, Grönke S, Siibak T, Stewart JB, Baggio F, Dols J, Partridge L, Falkenberg M, Wredenberg A, Larsson NG. Complementation between polymerase- and exonuclease-deficient mitochondrial DNA polymerase mutants in genomically engineered flies. Nat Commun. 2015. PMID: 26554610

Hensen F, Moretton A, van Esveld S, Farge G, Spelbrink JN. The mitochondrial outer-membrane location of the EXD2 exonuclease contradicts its direct role in nuclear DNA repair. Sci Rep. 2018. PMID: 29599527

Kauppila TES, Kauppila JHK, Larsson NG. Mammalian Mitochondria and Aging: An Update. Cell Metab. 2017. PMID: 28094012

Kukat A, Trifunovic A. Somatic mtDNA mutations and aging--facts and fancies. Exp Gerontol. 2009. PMID: 18585880

Matic S, Jiang M, Nicholls TJ, Uhler JP, Dirksen-Schwanenland C, Polosa PL, Simard ML, Li X, Atanassov I, Rackham O, Filipovska A, Stewart JB, Falkenberg M, Larsson NG, Milenkovic D. Mice lacking the mitochondrial exonuclease MGME1 accumulate mtDNA deletions without developing progeria. Nat Commun. 2018. PMID: 29572490

Medeiros TC, Thomas RL, Ghillebert R, Graef M. Autophagy balances mtDNA synthesis and degradation by DNA polymerase POLG during starvation. J Cell Biol. 2018. PMID: 29519802

Moretton A, Morel F, Macao B, Lachaume P, Ishak L, Lefebvre M, Garreau-Balandier I, Vernet P, Falkenberg M, Farge G. Selective mitochondrial DNA degradation following double-strand breaks. PLoS One. 2017. PMID: 28453550

Nicholls TJ, Zsurka G, Peeva V, Schöler S, Szczesny RJ, Cysewski D, Reyes A, Kornblum C, Sciacco M, Moggio M, Dziembowski A, Kunz WS, Minczuk M. Linear mtDNA fragments and unusual mtDNA rearrangements associated with pathological deficiency of MGME1 exonuclease. Hum Mol Genet. 2014. PMID: 24986917

Peeva V, Blei D, Trombly G, Corsi S, Szukszto MJ, Rebelo-Guiomar P, Gammage PA, Kudin AP, Becker C, Altmüller J, Minczuk M, Zsurka G, Kunz WS. Linear mitochondrial DNA is rapidly degraded by components of the replication machinery. Nat Commun. 2018. PMID: 29712893

Trifunovic A, Wredenberg A, Falkenberg M, Spelbrink JN, Rovio AT, Bruder CE, Bohlooly-Y M, Gidlöf S, Oldfors A, Wibom R, Törnell J, Jacobs HT, Larsson NG. Premature ageing in mice expressing defective mitochondrial DNA polymerase. Nature. 2004. PMID: 15164064

Yu Z, O'Farrell PH, Yakubovich N, DeLuca SZ. The Mitochondrial DNA Polymerase Promotes Elimination of Paternal Mitochondrial Genomes. Curr Biol. 2017. PMID: 28318978